Species Identity Dominates over Environment in Driving Bacterial Community Assembly in Wild Invasive Leaf Miners

ABSTRACT The microbiota of invasive animal species may be pivotal to their adaptation and spread, yet the processes driving the assembly and potential sources of host-microbiota remain poorly understood. Here, we characterized microbiota of four Liriomyza leaf miner fly species totaling 310 individuals across 43 geographical populations in China and assessed whether the microbiota of the wild leaf miner was acquired from the soil microbiota or the host plant microbiota, using high-throughput 16S rRNA sequencing. Bacterial communities differed significantly among four leaf miner species but did not mirror host phylogeny. Microbiota diversity in the native L. chinensis was significantly higher than in three invasive leaf miners (i.e., L. trifolii, L. huidobrensis, and L. sativae), yet the microbial community of the invasive species exhibited a more connected and complex network structure. Structural equation models revealed that host species identity was more important than environmental factors (e.g., geography, climate, or plants) in shaping microbiota composition. Using neutral and null model analyses, we found that deterministic processes like variable selection played a primary role in driving microbial community assembly, with some influence by stochastic processes like drift. The relative degree of these processes governing microbiota was likely correlated with host species but independent of either geographical or climatic factors. Finally, source tracking analysis showed that leaf miners might acquire microbes from their host plant rather than the soil. Our results provide a robust assessment of the ecological processes governing bacterial community assembly and potential sources of microbes in invasive leaf miners. IMPORTANCE The invasion of foreign species, including leaf miners, is a major threat to world biota. Host-associated microbiota may facilitate host adaption and expansion in a variety of ways. Thus, understanding the processes that drive leaf miner microbiota assembly is imperative for better management of invasive species. However, how microbial communities assemble during the leaf miner invasions and how predictable the processes remain unexplored. This work quantitatively deciphers the relative importance of deterministic process and stochastic process in governing the assembly of four leaf miner microbiotas and identifies potential sources of leaf miner-colonizing microbes from the soil-plant-leaf miner continuum. Our study provides new insights into the mechanisms underlying the drive of leaf miner microbiota assembly.

design was carefully carried out, and Materials & Methods of the article are described in detail. However, several concerns needed to be addressed before publication.
Major issues: 1. In Fig.1B, according to the illustration, grey represents L. trifolii. However, according to the figure, purple refers to L. trifolii. 2. "Reads for the endosymbiont Wolbachia (OTU9) were enriched in L. huidobrensis and present in the other two invasive leaf miners but not recovered from the native L. chinensis (Supplementary Fig. 2 and 3).". I didn't see the relevance between Wolbachia and gut microbiome. More details need to be added. 3. Fig.2 suggested that host species was the most important driver in governing the bacterial compositions in the leaf miners. However, to me, it is still unclear that the correlation between climate (geography/host plant) and leaf miner bacterial communities. Need to be clear somehow. 4. "PCoA showed that larval and adult leaf miners microbiota formed a close cluster, which were distinct from those found in leaves, roots and soil samples (ADONIS: R = 0.83, p < 0.001) (Fig. 6B)." However, given larvae, adults and leaves are close in Fig 6B, it's unfair to say 'distinct from those found in leave'. 5. There are many spelling errors throughout paper, please doublecheck accordingly. I listed few of them below. 6. Fig. 3 B -D, these pictures are inconsistent with figure legends (line 735). 7. Fig. 6 A and D, it is more harmonious to keep the same group colors in the two images.
Reviewer #2 (Comments for the Author): The manuscript entitled "Species identity dominates over environment in driving bacterial community assembly in wild invasive leaf miners" present findings from a survey of the bacterial communities associated with four leaf miner species, totaling 310 individuals across 43 geographical populations in China, and the continuum of soil -plants -leaf miner. The findings are interesting in that they appear to be relatively novel in the scope of animal microbiome, and provide insights into the patterns of bacterial assembly and microbial source tracking. Main conclusion in the study is that species identity dominates over environment in driving bacterial community assembly in wild leaf miners. This is a really great study and I'm happy the authors are reporting it.
Methods need a bit of clarification. I have a couple concerns about the method associated with bioinformatics analysis that can be cleared up relatively easily. Reviewers may want more detail on the bioinformatics pipelines for data filtering and taxon assignment.
A 'IMPORTANCE' Section is required.
Specific Comments: Line 151: A space is required to add between 'at' and '26{degree sign}C'. Line 155: These are whole insect extractions, rather than just gut? The author should clarify the methods they used. Line 164: Did you also run 'blank' samples as negative controls? Line 172: There is no mention of the how the library prep was done. Line 178: Citation for this? Line 182: Were analyses performed on rarefied samples or was another method of sequence depth standardization used? Line 188: perMANOVA Line 222-239: The rationale for the neutral and null models appears to be similar. Many readers will not be familiar the distinctions because they are similar so I suggest providing clear reasons for doing each model. This same thing applies to most of the other models, I would put into easily understandable language the reason you run each of these models/tests. Line 311: Please mention the name of the neutral model you use and briefly explain it already here. Line 465: This sounded like you provided a comprehensive overview of all leaf mining flies which I think number in the thousands.
The discussion section is thorough and interesting. Fig. 1-Bacterial taxonomy at the phylum level is not particularly informative. Figures at the class level could provide additional insights into potential functional groups present in the leaf miners. Fig. 2-This is a good figure.
Staff Comments:

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Reviewer comments: Reviewer #1 (Comments for the Author):
The assembly process of microbial communities is a long-standing interest in the field of microbial ecology. In particular, the driving forces shape intestinal microbiome are critical to decipher the host-microbe interactions. Herein, Zhu and co-authors worked on the microbiota of one native leaf miner L. chinensis and three close-related invasive species L. trifolii, L. huidobrensis, and L. sativae. By using structural equation models, they qualified host species identity was more important than environmental factors. Subsequently, they found variable selection was important to drive microbial community assembly through neutral and null model analysis. Further source tracking analysis showed that leaf miners might acquire microbes from host plants rather than the surrounding soil. In general, the work represents some interesting and informative results to the field, experimental design was carefully carried out, and Materials & Methods of the article are described in detail. However, several concerns needed to be addressed before publication. method associated with bioinformatics analysis that can be cleared up relatively easily.
Reviewers may want more detail on the bioinformatics pipelines for data filtering and taxon assignment.

Response:
To clarify our methods, we added the sentences in line 391-392: The raw sequence data were processed as described previously (24).
In line 392-395, we stated the bioinformatics pipelines for taxon assignment. Please check it.
Point 2: A 'IMPORTANCE' Section is required.

Response:
We added the following section in line 42-52:

IMPORTANCE
The invasion of foreign species, including leaf miners, is a major threat to world biota.
Host-associated microbiota may facilitate host adaption and expansion in a variety of ways. Thus, understanding the processes that drive leaf miner microbiota assembly is imperative for better management of invasive species. However, how microbial communities assemble during the leaf miner invasions, and how predictable the processes remain unexplored. This work quantitatively deciphers the relative importance of deterministic process and stochastic process in governing the assembly of four leaf miner microbiotas and identifies potential sources of leaf miner-colonizing microbes from the soil-plant-leaf miner continuum. Our study provides new insights into the mechanisms underlying the drive of leaf miner microbiota assembly.